Oligonucleotides, oligonucleotide analogs and other sequence-specific binding polymers designed to block translation of selected messenger RNA (the sense strand) are commonly called antisense oligonucleotides. Development of such oligonucleotides, for therapeutic applications entails selecting a target genetic sequence unique and critical to the pathogen or pathogenic state one wishes to treat. One then assembles an oligomer of genetic bases (adenine, cytosine, guanine, and thymine or uracil) complementary to that selected sequence. When such an antisense oligonucleotide binds to its targeted disease-causing sequence, it can inactivate that target and thereby alleviate the disease. Antisense oligonucleotides offer the prospect of safe and effective therapeutics for a broad range of intractable diseases. Nonetheless, developing therapeutics that function by a true antisense mechanism presents a number of forbidding challenges. The oligonucleotides should achieve adequate efficacy at a concentration attainable within the cells of the patient. They should inhibit their selected target sequences without concomitant attack on any other sequences in the patient's pool of approximately 200 million bases of unique-sequence RNA. They should be stable in extracellular compartments and within cells. They must be deliverable into the cellular compartments containing their targeted sequences. They should be adequately soluble in aqueous solution. Finally, they should exhibit little or no toxicity at therapeutic concentrations.
First-generation antisense oligonucleotides comprised natural genetic material (Belikova et al. (1967) Tetrahedron Lett. 37, 3557-3562; Zamecnik et al. (1978) Proc. Natl. Acad. Sci. USA 75, 280-284; Summerton (1979) J. Theor. Biol. 78, 77-99) and often contained crosslinking agents for binding their targets irreversibly (Summerton et al. (1978) J. Mol. Biol. 122, 145-162). As the design challenges became more fully appreciated, a number of non-natural antisense structural types were developed in an effort to improve efficacy, stability and delivery. Of particular note are the early non-ionic DNA analogs including phosphotriester-linked DNA and methylphosphonate-linked DNA (Cohen (1989) Oligodeoxynucleotides: Antisense Inhibitors of Gene Expression, CRC Press, pp. 82-92). Other nucleic acid analogs of note include carbamate-linked DNA (Cohen (1989) Ohgodeoxynucleotides: Antisense Inhibitors of Gene Expression, CRC Press, pp. 97-117), phosphoroamidate-linked DNA (Froehler et al. (1988) Nucleic Acids Res. 16, 4831-4839) and 2′-O-methyl RNA (Shibahara et al. (1989) Nucleic Acids Res. 17, 239-252). These second generation oligonucleotides include oligonucleotides containing acyclic backbone moieties, including nylon (Weller et al. (1991) J. Org. Chem. 56, 6000-6006; Huang et al. (1991) J. Org. Chem. 56, 6007-6018), the exceptionally high-affinity peptide nucleic acids (PNA) (Egholm et al. (1992) J. Am. Chem. Soc. 114, 1895-1897) and related types (U.S. Pat. No. 5,217,866).
One approach to improving the potency of antisense oligonucleotides is to enhance the affinity or the efficiency with which the antisense oligonucleotides interact with their targets and induce RNase degradation of their target gene transcripts. The doses at which effects have been observed generally range from 10 to 30 mg/kg i.v. (Miraglia et al. (2000) Antisense Nuc. Acid Drug Devel. 10, 453-461). Some clinical studies, however, have not demonstrated antisense activity at doses up to 30 mg/kg i.v. (Rudin et al. (2001) Clin. Cancer Res. 7, 1214-1220; Kushner et al. (2000) Curr. Oncol. Reports 2, 23-30), indicating that results vary based on the structure of the oligonucleotide administered. Typical dose-response curves for antisense oligonucleotides both in vivo and in vitro, often reveal that less than a factor of ten often separates the concentration producing antisense activity from the concentration producing no activity (Branch (1998) Trends Biochem. Sci. 23, 45-50). Since the ratio of antisense to non-antisense effects drops sharply outside a restricted concentration range, it remains challenging to identify common structural features for any antisense oligonucleotide that will enhance affinity and efficiency of the oligonucleotide for its target. Furthermore, no studies to date have identified common structural features of antisense oligonucleotides that would make them suitable for oral administration, thus necessitating intravenous administration (Chen et al. (2000) Antisense Nuc. Acid. Drug Develop. 10, 415-422). Identification of common structural modifications of antisense oligonucleotides that facilitate oral or topical administration would therefore also be advantageous.
Although each of these newer structural types provides one or more significant advantages over the first-generation oligonucleotides, none yet appear to provide the full combination of properties needed in antisense therapeutics for successful therapeutic applications.